Immunoglobulins are glycoproteins which exist in serum, and tissue and body fluid of all mammals and have a function of recognizing foreign antigens (Non-patent Literature 1). The antibody is involved in biological defense through activation of the complement system and activation of their effector functions, such as effects of cell phagocytosis, antibody-dependent cellular cytotoxicity, mediator release, antigen presentation, via receptors (FcR) existing on the cell surface. There are five different classes of human immunoglobulins, namely IgG, IgA, IgM, IgD, and IgE. IgG can be classified into the 4 subclasses of IgG1, IgG2, IgG3, and IgG4. Also, IgA can be classified into 2 subclasses of IgA1 and IgA2. The basic structure of immunoglobulin is composed of two L (light) chains and two H (heavy) chains. Class and subclass of the immunoglobulin are determined by H chains. Different class and subclass of immunoglobulins are known to have different functions. For instance, the level of complement-binding ability decreases in the following order: IgM>IgG3>IgG1>IgG2. Also, the level of affinity to FcγRI (Fc receptor I) decreases in the following order: IgG3>IgG1>IgG4>IgG2. In addition, IgG1, IgG2, and IgG4 can bind to protein A. Specificity of antibody to an antigen is determined by the combination of heavy chains and light chains. In the case of IgG, one molecule is composed of two pair of a heavy chain and a light chain and has two antibody recognition sites per one antibody molecule.
In recent years, many clinical trials for monoclonal antibodies have been carried out. In addition, there are many monoclonal antibodies which are commercially available (Non-Patent Literature 2). In 1986, a mouse anti-CD3 antibody, muromonab-CD3, was approved by the FDA. In 1994, a chimeric antibody abciximab in which a mouse constant region of the antibody was substituted with a human constant region in order to reduce the antigenicity was approved. Humanization technologies were developed in order to further reduce the antigenicity. In 1997, an anti-CD20 humanized antibody, daclizumab, in which a variable region was humanized was approved. In 2002, the fully human anti-TNFα antibody, adalimumab, was approved.
For the purpose of modifying the antibody activity, production of mutants of various antibodies has been attempted. For example, a multivalent antibody which recognizes different antigens was produced by a hybrid hybridoma. However, when this method is used, since two different types of heavy chain and light chain are expressed in one cell, approximately ten combinations of the heavy chain and the light chain of an antibody are obtained. Accordingly, as a result, the productivity of a multivalent antibody having desired combination of a heavy chain and a light chain is lowered, and moreover it is difficult to isolate and purify the targeted multivalent antibody (Non-Patent Literature 3).
In order to overcome this problem, an attempt to produce an antibody having a correct combination of polypeptides by reducing the variations of the combinations of subunits by linking multiple antigen recognition sites in one polypeptide chain has been reported. For example, an antibody comprising scFv in which antigen recognition sites of a heavy chain and a light chain are linked to each other via one polypeptide (Non-Patent Literature 4) is known. Moreover, an antibody in which two antigen recognition sites are linked each other using an CH1 domain of a H chain constant region, a partial fragment of thereof, or L chain constant region of an antibody IgG1, or a flexible linker (Gly-Gly-Gly-Gly-Ser; SEQ ID NO: 376) (Non-Patent Literature 5, Patent Literature 1, Patent Literature 2) or the like has been reported.
However, such a conventional multivalent antibody has disadvantages in that antibody protein is easily aggregated, and stability and productivity thereof are low.